The plasma membrane Na-H exchanger NHE1 has an established role in intracellular pH homeostasis. Work during the past funding period revealed that NHE1 also acts as an anchor for actin filaments by binding directly the ERM family of actin-binding proteins. In fibroblasts, ERM binding retains the localization of NHE1 at the leading edge of lamellipodia and is necessary for the assembly of actin filaments in membrane protrusions. The coordinate functions of NHE1 in ion translocation and actin anchoring are necessary for directed cell migration and are required for cell polarity, actin polymerization, and remodeling of focal adhesions. A novel DNHE1 in Drosophila that binds an ERM ortholog, and a novel DdNHE1 in Dictyostelium discoideum that is necessary for cell polarity and chemotaxis were identified, suggesting that actin anchoring by NHE1 and a requirement for NHE1 in directional cell movement are evolutionarily conserved. The objective of the current proposal is to determine how NHE1 regulates directional cell movement by investigating the hypothesis that its two functions act coordinately to generate an asymmetrically localized H+ efflux that is necessary for maintaining polarity. Studies in Aim 1 will determine how NHE1 regulates membrane protrusions and actin polymerization in mammalian fibroblasts by asking how localized H+ fluxes by NHE1 promote cell protrusion, the formation of actin free barbed ends, and the spatially restricted activation of Rac and Cdc42 at the leading edge of migrating cells. Whether NHE1 acts in a self-generating positive feedback loop at the leading edge of migrating cells will also be determined. Aim 2 addresses how the two functions of NHE1 in ion translocation and actin anchoring coordinately remodel cell-substrate adhesions in mammalian fibroblasts by asking whether actin anchoring by NHE1 is necessary for integrin activation or for subsequent steps in the assembly of focal adhesion-associated proteins, and whether ion translocation by NHE1 promotes focal adhesion disassembly through a pH dependent disruption of the integrin-cytoskeleton linkage. How newly formed focal adhesions contribute to the NHE1-dependent migratory phenotype will also be determined. Aim 3 focuses on why DdNHE1 is necessary for Dictyostelium chemotaxis by asking whether it generates localized H+ efflux, whether it maintains polarity by acting on signals at leading or trailing edges of the cell, and whether genetic screens identify DdNHE1 effectors mediating polarity and chemotaxis. Overall, this research plan addresses significant questions related to how polarity is developed and maintained and how spatially restricted signaling processes drive cell migration.